The structure and mechanisms of bone-implant integration, osteointegration, are poorly understood. Our long-term goal is to identify the biomechanical and molecular properties in bone-titanium integration and to develop improved implant technology for clinical applications. Using a differential display-polymerase chain reaction (DD-PCR) assay, we isolated and identified prolyl 4-hydroxylase alpha-subunit (P4Ha) as a gene transcript specifically upregulated in early stage of bone healing with titanium implants. P4H functions during post-translational hydroxylation of collagen chains, a process essential for cross-linking individual collagen molecules into a trimetric mature collagen to prevent intracellular degradation. The objective of this application is to identify the role of P4H in establishing tissue-titanium integration. We recently discovered that mineralized tissue cultured on titanium, especially acid-etched, roughened titanium, is harder than that cultured on a polystyrene surface. Our studies have also demonstrated characteristic collagen deposition dynamics on titanium. We hypothesize that early increased P4Ha gene expression in mineralized tissue on titanium is responsible for the observed collagen deposition dynamics and enhanced hardness and interfacial adhesion of the tissue on titanium. The following specific aims are formulated to test this thesis. Specific Aim 1: To establish the role of P4H on collagen deposition dynamics in the mineralized tissue. We will study loss-in-function by inhibiting P4H function with the hydroxylation inhibitor, 3,4 dehydroproline at early, mid and late stages of osteoblastic culture with and without titanium. Gain-in-function will be studied by transfecting the plasmid expression vector encoding P4Ha into osteoblasts at early and mid stages of culture. We will determine the density and localization of collagen deposition change in the various culture conditions. Changes in the expression of representative osteoblastic genes, cellular proliferation, mineralization and tissue surface morphology will also be examined. Specific Aim 2: To establish the role of P4H on the biomechanical properties of mineralized tissue and the relationship between hypoxia and P4H. We have shown that enhanced biomechanical property of mineralized tissue on titanium is associated with characteristic collagen-related structural changes. Therefore, we will control collagen synthesis using P4Ha manipulation and evaluate hardness, elastic modulus, critical load (force to delaminate tissue) and interface adhesion strength. Relationships between P4H function assessed by P4H inhibition or P4Ha gene transfection and the biomechanical properties and pattern in collagen deposition will be studied. We, further, will evaluate the effectiveness of P4H-driven biomechanical enhancement on titanium having different surface topographies. Finally, to address the regulatory function of HIF on P4H, we will establish the dissolved oxygen concentration in the culture with or without titanium, HIF expression levels, and the effect of HIF expression on P4H transcription. We seek to elucidate the mechanisms underlying tissue-titanium integration and the biomechanics of integrated mineralized tissue and anticipate that identifying a link with P4H will provide an opportunity to pursue P4Ha as a potential, novel strategy for implant therapy. [unreadable] [unreadable]